1a.Objectives (from AD-416):
To determine the identities of phytoplasma pathogens causing diseases of plants in Puerto Rico and to develop technologies for sensitive and accurate detection and classification of the phytoplasmas.
1b.Approach (from AD-416):
We will identify, characterize, and classify, according to 16S rDNA RFLP group and ‘Candidatus Phytoplasma’ species, phytoplasma strains in diseased plants in Puerto Rico. This information will be used by both parties to jointly develop tools and technologies for rapid and accurate detection and identification of these phytoplasmas. The Cooperator will investigate the occurrence, spread, prevalence, and severity of phytoplasmal diseases of plants in Puerto Rico.
In this reporting period, experimental protocols transferred to the Cooperator were established in the Cooperator laboratory and utilized for DNA extraction from symptomatic diseased plants in Puerto Rico and for determining the possible presence of phytoplasma in the plants. Polymerase chain reactions (PCRs) were used for the amplification of phytoplasma DNA from the ribosomal RNA operon, and amplified DNAs were subjected to restriction fragment length polymorphism analysis and nucleotide sequence analysis. The resulting data were used to determine the identities of phytoplasmas, through applications of the ARS laboratory’s publically accessible, online interactive phytoplasma classification program, iPhyClassifier. Through the use of these approaches, diseased plants belonging to no less than seven (7) species were found to be infected by phytoplasmas in Puerto Rico. The phytoplasma strains detected were preliminarily identified and classified. Further work was planned to confirm the identification and classification of the phytoplasma strains; to amplify, nucleotide sequence, and analyze the nucleotide sequences from non-ribosomal genes of the detected phytoplasmas; to identify molecular markers of the Puerto Rico phytoplasma strains; and to gain understanding of the evolutionary relationships of the detected phytoplasmas. The results from these and other experiments will aid the development of improved technologies for sensitive and accurate detection and classification of the phytoplasmas affecting plants, and will aid in the management of phytoplasmal diseases of important crops and ornamental plants in Puerto Rico and the continental U.S.